Method for preparing aminocyclohexylpenicillin

ABSTRACT

An enzymatic process for the preparation of aminocyclohexylpenicillin is disclosed. The process involves reacting together 6-aminopenicillanic acid or other penicillins and aminocyclohexyl carboxylic acid, or esters and/or acid addition salts thereof in the presence of an enzyme elaborated by such microorganisms as are found in the genera Pseudomonas, Kluyvera, Escherichia, Aerobacter, Micrococcus, Streptomyces, Nocardia, Aspergillus or Penicillium.

United States Patent [1 1 Nara et al.

[ 51 Feb. 20, 1973 [54] METHOD FOR PREPARING AMINOCYCLOHEXYLPENICILLIN[75] Inventors: Takashi Nara; Ryo Okachi, both of Tokyo, Japan [73]Assignee: Kyowa Hakko Kogyo Co., Ltd.,

Tokyo, Japan [22] Filed: Oct. 12, 1970 [211 App]. No.: 80,227

[30] Foreign Application Priority Data Oct. 24, 1969 Japan ..44/84653 s2U.S. Cl "195/36? 51 im. Cl. "cm 9/06 58 Field of Search ..l95/36 P [56]References Cited UNITED STATES PATENTS 3,047,467 7/1962 Doyle et al...l95/36 P 3,553,201 1/1971 Clark et al ..260/239.l

Primary Examiner-Alvin E. Tanenholtz Attorney-Ralph T. Lilore 5 7ABSTRACT 10 Claims, No Drawings METHOD FOR PREPARINGAMINOCYCLOHEXYLPENICILLIN BACKGROUND OF THE INVENTION This inventionrelates to the method for producing aminocyclohexylpenicillin. Moreparticularly it relates to the enzymatic production ofaminocyclohexylpenicillin from penicillins, especially.6-aminopenicillanic acid, in the presence of aminocyclohexylcarboxylicacid and esters and acid addition salts thereof.

Aminocyclohexylpenicillin is a broad spectrum-antibiotic effectiveagainst a wide range of gram negative and gram positive pathogenicbacteria. This penicillin is characteristic in being more active in vivothan in vitro to many bacterial organisms causing human infection. Thispoint is very different from ampicillin which is also a type of broadspectra in antibacterial properties but is inferior in vivo activitiesto aminocyclohexylpenicillin. Aminocyclohexylpenicillin can curediseases caused by Staphylococcus aureus, Diplacoccus pneumoniae,Streptococcus sp., Neisseria sp., Escherichia coli, Shigella sp. andmany others.

Dosage of aminocyclohexylpenicillin required for oral administration isabout 1 g. per day.

DETAILED EXPLANATION OF THE INVENTION A process has now been discoveredwhereby aminocyclohexylpenicillin can be elaborated from penicillins,especially 6-aminopenicillanic acid, (hereinafter referred to as 6-APA)as well as from other penicillins. The process involves reacting theaminocyclohexylcarboxylic acid or functional derivatives thereof withthe penicillins in the presence of an enzyme derived from microorganismsof any of the genera Pseudomonas, Kluyvera, Escherichia, Aerobacter,Micrococcus, Streptomyces, Nocardia, Aspergillus or Penicillium. As willbecome apparent below the actual cell bodies or culture liquid of themicroorganisms may be used as the reaction vehicle. Pseudomonas andKluyvera are the preferred genera.

One of the basic substrates utilized in the process of the invention isa penicillin compound, that is a compound having the basic structure oresters, salts and acid addition salts thereof, wherein R is hydrogen oran acyl radical normally found in the penicillin compounds. R is usuallya pharmaceutically acceptable group such as is found in penicillin V, Gand the like though it need not be. Typical groups are 5 phenoxyacetyl,phenylacetyl, 'y-chlorocrotyl mercaptoacetyl and the like. Virtually anygroup can be used, the only criteria being that the basic penicillinskeletal structure be present. Preferred for use is 6-APA or,

penicillin G or V. Penicillin V is phenoxymethylpenicil- 10 lin andpenicillin G is benzylpenicillin.

The penicillin substrate is preferably employed as a salt, usually analkali metal or alkaline earth metal salt and preferably as the sodium,potassium or calcium salt. The compounds may also be used in the form of15 the acid addition salts such as the hydrochloride, hydrobromide,hydroiodide, hydrogen phosphate or the like.

The aminocyclohexylcarboxylic acid reactant may be employed as the freeacid but it is preferred to utilize 20 the same as a functionalderivative and most preferably as an acid addition salt of thederivative. Suitable acid derivatives are the amides, alkyl esters oralkali metal salts of the compound. Preferred for use are the alkylesters having from one to four carbon atoms and espe- 5 cially themethyl or ethyl ester, although the sodium or potassium salts may alsobe used. As acid addition salts there may be employed the acid saltsderived from the mineral acids such as the hydrochloride, as ispreferred, or the hydrobromide, hydroiodide, hydrogen phosphate or thehydrogen sulfate compounds. The following flow diagrams arerepresentative of the process of the invention.

a. In case 6-APA is used as a substrate;

l-aminocyclohexylfi-aminopenicillanic acid (ti-APA) carboxylic acid b.In case penicillin G is used as a substrate:

phonylacctic acid aininocyclohcxylpenicillin[6-(1-nm1noeyclohcxanc.carboxarnide) pcnicillanic acid] Themicroorganisms used in the present invention are those belonging toPseudomonas, Kluyvera, Escherichia, Aerobacter, Micrococcus,Streptomyces, Nocardia, Aspergillus or Penicillium, especially thestrains thereof which have acquired a very powerful enzymatic activityfor synthesizing aminocyclohexylpenicillin by applying a hereditarymutation (for example, ultraviolet ray irradiation, X-ray irradiation ortreatment with amutation-initiating agent) thereto.

The specific bacteria, ray fungi and molds belonging to said genera andused in the present invention, have a very powerful ability to formaminocyclohexyl-penicillin from 6-APA or other penicillins andaminocyclohexylcarboxylic acids. The optimum pH of the enzymaticactivity in these microorganisms lies in a relatively wide rangespanning slight acidity to neutrality to slight alkalinity.

To obtain the enzyme used in the process of the invention, strains ofthese microorganisms are cultured so that the desired enzyme is producedwithin the cell bodies and also preferably within the culture liquor.Culturing is preferably carried out for a sufficient time to allow theextracellular production and accumulation of the enzyme as describedbelow.

As a culturing medium for these strains, there is employed a mediumcontaining a carbon or energy source such as giucose, sucrose, starch,molasses, sorbitol, or other natural products containing an organicnitrogen source as well. For example, peptone, meat extract, yeastextract, cornsteep liquor, and the like may be used. The medium furthercontains suitable amounts of a nitrogen source such as urea, ammonia,ammonium sulfate, and ammonium chloride; or a natural nitrogen sourcesuch as cornsteep liquor, peptone, meat extract, yeast extract, and thelike. Inorganic salts such as phosphates (potassium primary phosphate,potassium secondary phosphate, and the like); magnesium salts such asmagnesium sulfate; metal ions such as iron, sodium, potassium manganese,zinc, calcium and the like and anions such as chlorides and nitrates arealso preferably present. Other further nutrient substances necessary forthe growth of these strains are employed as needed.

Culturing is carried out at a culturing temperature of 20-50 C. and a pHof 5.0-9.0 under aerobic conditions such as by shake-culturing orsubmerged culturing techniques with aeration and agitation. Culturingtime is usually 1 to 7 days. In this manner, the enzyme used for thesynthesis is formed in the cultured cell bodies and usually within theculture liquor as well.

As indicated previously, the source of the enzyme used in the process ofthe invention may be the culture liquor itself, the cell bodies, theculture liquor free from the cell bodies, or the free enzyme alone,purified by salting-out with ammonium sulfate, dialysis, precipitationwith acetone or ethanol, column chromatography, or the like. When thecell bodies are used it is preferred that they be used in a suspensionor in an aceton-dried state. When the culture liquor is used, thesubstrate is added to the culture liquor and subjected to reactiontherewith after the pH has been adjusted as indicated below.

The enzymatic reaction is carried out in a reaction liquid containingthe two substrates and a suitable amount of the enzyme. The reactionmedium may well be the fermentation liquor itself to which thesubstrates have been added. A buffer solution is preferably added to thereaction medium to facilitate the reaction within an optimum pH range.The reaction is possible in a wide pH range of 3-8, but particularlysuitable is a range of from 4.5-7.5. The reaction is suitably carriedout at 25 50 C., but preferably at 30 38 C. for l to 24 hours.

After the completion of the reaction, the aminocyclohexylpenicillin maybe isolated and recovered by such well-known methods in suitablecombination as a transfer extraction, precipitation via the addition ofan organic solvent, or via isoelectric point precipitation.Additionally, ion exchange, column chromatography, and the like may beemployed for isolation purposes.

The aminocyclohexylpenicillin obtained in the process of the inventionis useable as an effective drug that can cure diseases caused byStaphylococcus aureus, Diplocaccus pneumoniae, Streptococcus sp.,Neisseria sp., Escherichia coli, Shigella sp. and many others.

Dosage of aminocyclohexylpenicillin required for oral administration isabout 1 g. per day.

The following examples are given to illustrate preferred embodiments ofthe present invention.

EXAMPLE 1 The microorganism, Pseudomonas melanogenum ATCC 17808 is used,as a seed microorganism and a medium containing 1 percent peptone, 1percent meat extract, 0.5 percent yeast extract and 0.3 percent sodiumchloride is used as the seed medium. One platinum loop of the seedmicroorganism is inoculated into 20 ml. of the medium in a 250-ml.Erlenmeyer flask and culture at 30 C. for 24 hours with shaking.Thereafter 2 ml. of the seed culture liquor is inoculated in 20 ml. of amain fermentation medium placed in a 250-ml. Erlenmeyer flask.

Composition of the main fermentation medium is as follows: 0.5 percentpeptone, 0.5 percent yeast extract, 0.5 percent Na L-glutamate, and 0.3percent sodium chloride; pH before sterilization: 7.3 (adjusted with 5 NNaOH).

After 48 hours from the start of the shaking culture at 30 C., cellbodies are separated from the fermentation liquor by centrifugalprecipitation, and washed twice with an aqueous 0.9 percent sodiumchloride solution. The cell bodies are then suspended in a 1/30 Mphosphoric acid buffer of pH 6.8 at a dry cell content level of 10mg./ml.. To this suspention are added sufficient 6-APA and themethylester of aminocyclohexylcarboxylic acid hydrochloride to obtain alevel of 1.5 mg./ml. of the former and 5 mg./ml. of the latterrespectively. The reaction is then carried out at 35 C. for 4 hours toyield 1.35 mg./ml. of aminocyclohexylpenicillin formed in the reactionsolution.

EXAMPLE 2 As a seed microorganism, Kluyvera citrophila ATCC 21285 isused The culturing conditions are the same as in Example 1, but a mainfermentation medium prepared by adding 0.2 percent phenylacetic acid tothe medium of Example 1 is used. The reaction is carried out in the samemanner as in Example 1, using the cell bodies obtained after 48 hoursfrom the start of culturing but using 3 mgJml. of potassium salt ofpenicillin G to the reaction solution of Example 1 in place of 6-APA.Further, aminocyclohexylcarboxylic acid amide hydrochloride is used inplace of the methyl ester of aminocyclohexylcarboxylic acidhydrochloride. In this manner, 2.5 mg./ml. of aminocyclohexylpenicillinis formed in the reaction solution after 5 hours from the start of thereaction.

EXAMPLE 3 Escherichia coli ATCC 13281 and Aerobacter aerogenes ATCC 8308are used as seed microorganisms in two separate runs. The culturingconditions are the same as in Example 1. After 48 hours from the startof culturing, the cell bodies are recovered, suspended in an aqueousmedium as described in Example 1, and then poured into a large amount ofacetone. After aspiration filtering, acetone-dried cell bodies areobtained by ether washing the filtered cells.

The enzymatic reaction is carried out under the same conditions as inExample 1, using the acetone-dried cell bodies as the source of theenzyme. An additional 0.5 mg./ml. of 6-APA is added to the reactionsolution after 3 hours from the start of the reaction and the reactionis continued for a further 2 hours. In this manner, the amount ofaminocyclohexylpenicillin accumulated after 5 hours from the addition ofthe substrate corresponds to 1.20 mg./ml. in the case of Escherichiacoli and l.l8 mg./ml. in the case of Aerobacter aerogenes.

EXAMPLE 4 Two strains of Streptomyces phaeochromogenes ATCC 21289 andNocardia globerula ATCC 21292 are used as seed microorganisms in twoseparate runs. A medium containing 3 percent soluble starch, 2 percentsoybean powders, 0.5 percent yeast extract and 0.1 percent calciumcarbonate at a pH of 7.3 (before sterilization) is used as the mainfermentation medium. Other culturing conditions are the same as inExample 1. At the fourth day of the culturing, 2.5 mg./ml. of penicillin(penicillin G in the case of Streptomyces phaeochromogenes andpenicillin V in the case of Nocardia globerula) and 10 mg./ml. of theethyl ester of aminocyclohexylcarboxylic acid sulfate are added thereto.The pH of the fermentation liquor is adjusted to 6.8, and thefermentation is further continued. During fermentation, the pH of thefermentation liquor is adjustedto 6.8 at 1 hour intervals withhydrochloric acid or a sodium hydroxide solution. In this manner, theamounts of aminocyclohexylpenicillin accumulated in the fermentationliquor after 6 hours from the addition of the substrates corresponds to1.12 mg./ml. in the case of Streptomyces phaeochromogenes and 1.85mg./ml. in the case of Nocardia globerula.

EXAMPLE 5 Two strains of Aspergillus oryzae ATCC 16450 and Penicilliumchrysogenum ATCC 10135 are used as seed microorganisms in two separateruns. A medium containing 3 percent sucrose, 0.2 percent sodium nitrate,0.1 percent potassium secondary phosphate, 0.05 percent magnesiumsulfate, 0.05 percent potassium chloride, and 0.001 percent ferroussulfate at a pH of 6.8 before sterilization is used as the mainfermentation medium. 50 ml. of the main fermentation medium is placed ina 500-ml. Sakaguchi flask, and one platinum loop of the seedmicroorganism is inoculated therein. On the fifth day after shaking theculture at 27 C., 6- APA and potassium aminocyclohexylcarboxylate areadded to the fermentation liquor so that the content of each correspondsto 2.0 mg./ml. and 10 mg./ml., respectively. The pH of the fermentationliquor is then adjusted to 5.0 and the fermentation is furthercontinued. The amounts of aminocyclohexylpenicillin accumulated in thefermentation liquor after 6 days from the start of culturing are 2.02mg./ml. in the case of Aspergillus oryzae, and 2.31 mg./ml. in the caseof Penicillium chrysogenum, respectively.

After the separation of mycelia, about 2 l. of the fermentation liquoris passed through a column packed with 50 ml. of Na form of Dowex 50 W X4 (a strongly acidic cation exchange resin available from the DowChemical Co., U.S.A.) bufferized with 0.2 M-pH 2.0- citrate buffer priorto use. The product adsorbed on the resin is then eluted with 0.2 M-pH4.0- citrate buffer and then with 0.2 M-pH 7.0-citrate buffer. Pooledfractions containing the penicillin are adsorbed on active carbon (1.51.) and then eluted with percent methanol. The eluate is concentrated invacuo at 40 C. Adding aceton slowly to the concentrate, crude crystalsof the aimed product are obtained. The yield is 2.3 g.

We claim:

1. A method for preparing aminocyclohexylpenicillin, which comprisesreacting a penicillin with a compound selected from the group consistingof aminocyclohexylcarboxylic acid, and functionally equivalentderivatives thereof in the presence of an aminocyclohexyl-penicillinproducing enzyme derived from the culturing of a microorganism belongingto the genera Pseudomonas, Kluyvera, Escherichia, Aerobacter,Micrococcus, Streptomyces, Nocardia, Aspergillus or Penicillium.

2. The method according to claim 1 wherein the penicillin is6-aminopenicillanic acid, its alkylesters, alkali metal, alkaline earthmetal salts or acid addition salts thereof.

3. The method according to claim 1 wherein the penicillin is PenicillinG.

4. The method according to claim 1 wherein the penicillin is PenicillinV.

5. The method according to claim 1 wherein the reaction is carried outat a pH of from 3 to 8 and at a temperature of from 25 to 50 C.

6. The method according to claim 5 wherein the pH is from 4.5 to 7.5 andthe temperature is from 30 to 3 8 C.

7. The method according to claim 5 wherein the reaction is carried outfor from 1 to 24 hours.

8. The method according to claim 1 wherein the enzyme is produced byaerobically culturing the microorganism in a nutrient medium at atemperature of from 20 to 50 C. and a pH offrom 5 to 9.

9. The method according to claim 1 wherein the enzyme is contained inthe cell bodies of said microorganisms and the reaction is carried outin the presence of said cell bodies.

10. The method according to claim 1 wherein the enzyme is present in aculture liquor obtained from said microorganisms and the reaction iscarried out in the presence of said culture liquor.

1. A method for preparing aminocyclohexylpenicillin, which comprisesreacting a penicillin with a compound selected from the group consistingof aminocyclohexylcarboxylic acid, and functionally equivalentderivatives thereof in the presence of an aminocyclohexyl-penicillinproducing enzyme derived from the culturing of a microorganism belongingto the genera Pseudomonas, Kluyvera, Escherichia, Aerobacter,Micrococcus, Streptomyces, Nocardia, Aspergillus or Penicillium.
 2. Themethod according to claim 1 wherein the penicillin is6-aminopenicillanic acid, its alkylesters, alkali metal, alkaline earthmetal salts or acid addition salts thereof.
 3. The method according toclaim 1 wherein the penicillin is Penicillin G.
 4. The method accordingto claim 1 wherein the penicillin is Penicillin V.
 5. The methodaccording to claim 1 wherein the reaction is carried out at a pH of from3 to 8 and at a temperature of from 25* to 50* C.
 6. The methodaccording to claim 5 wherein the pH is from 4.5 to 7.5 and thetemperature is from 30* to 38* C.
 7. The method according to claim 5wherein the reaction is carried out for from 1 to 24 hours.
 8. Themethod according to claim 1 wherein the enzyme is produced byaerobically culturing the microorganism in a nutrient medium at atemperature of from 20* to 50* C. and a pH of from 5 to
 9. 9. The methodaccording to claim 1 wherein the enzyme is contained in the cell bodiesof said microorganisms and the reaction is carried out in the presenceof said cell bodies.